GlmU polypeptide and DNA encoding the polypeptide

ABSTRACT

The present invention relates to a polypeptide having the activity of N-acetylglucosamine-1-phosphate uridyltransferase (hereinafter referred to as GlmU), a DNA coding for the polypeptide, a recombinant DNA containing the DNA, a transformant carrying the recombinant DNA, a method of culturing the transformant for producing the GlmU polypeptide, and a method of culturing the transformant for producing uridine 5′-diphosphate-N-acetylglucosamine. 
     According to the present invention, mass-scale production of the GlmU polypeptide derived from microorganisms belonging to the genus  Corynebacterium glutamicum  has been enabled by genetic recombinant technology. By using the enzyme, uridine 5′-diphosphate-N-acetylglucosamine can be produced efficiently.

TECHNICAL FIELD

The present invention relates to a polypeptide having the activity ofN-acetylglucosamine-1-phosphate uridyltransferase (hereinafter referredto as GlmU), a DNA coding for the polypeptide, a recombinant DNAcontaining the DNA, a transformant carrying the recombinant DNA, amethod for producing the GlmU polypeptide by using the transformant, anda method for producing uridine 5′-diphosphate-N-acetylglucosamine byusing the transformant.

BACKGROUND ART

N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an enzymethat catalyzes the production of uridine5′-diphosphate-N-acetylglucosamine (hereinafter referred to asUDP-GlcNAc), an intermediate in biosynthesis of lipo-polysaccharides ofgram-negative bacteria. Regarding GlmU polypeptides, the enzyme ispurified from Escherichia coli, and it has been clarified that theenzyme catalyzes uridylation and also N-acetylation ofglucosamine-1-phosphate [J. Bacteriol., 176, 6852 (1994)].

The glmU gene is obtained from bacteria of the genus Escherichia [J.Bacteriol., 175, 6150(1993)], those of the genus Bacillus [J.Bacteriol., 174, 6852 (1992)], those of the genus Streptococcus(Japanese Published Unexamined Patent Application No. 155582/1999), andthose of the genus Neisseria [J. Bacteriol., 177, 6902 (1995)], but theglmU gene is not identified in bacteria of the genus Corynebacterium.

It is reported that the N-acetylation activity of GlmU derived fromEscherichia coli is unstable [J. Bacteriol., 176, 5788 (1994)], and theindustrial application of the GlmU is problematic.

Regarding other bacteria from which the genes have been isolated, thereis no description that suggests industrial production of GlmUpolypeptides by the use of the gene.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a polypeptide havingGlmU activity, a DNA coding for the polypeptide, a method for producingthe polypeptide having GlmU activity by using the DNA, and an industrialmethod for producing UDP-GlcNAc by using the polypeptide.

The present inventors have intensively studied to attain the object asmentioned above, and have found that a gene which complements thetemperature sensitivity of the lysozyme-sensitive strain ofCorynebacterium glutamicum codes for the polypeptide having GlmUactivity, and on the basis of this finding, we have completed thepresent invention.

That is, the present invention relates to the following (1) to (13):

-   -   (1) A polypeptide comprising the amino acid sequence represented        by SEQ ID NO: 1.

(2) A polypeptide comprising an amino acid sequence, in which one ormore amino acids have been deleted, substituted or added in the aminoacid sequence represented by SEQ ID NO: 1, and havingN-acetylglucosamine-1-phosphate uridyltransferase activity.

The polypeptide comprising the amino acid sequence represented by of SEQID NO: 1 in which one or more amino acids have been deleted, substitutedor added, and having N-acetylglucosamine-1-phosphate uridyltransferaseactivity is prepared according to the site-directed mutagenesisdescribed in Molecular Cloning, A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press (1989) (this is hereinafter referredto as Molecular Cloning, 2nd Ed.), Current Protocols in MolecularBiology, John Wiley & Sons (1987-1997) (this is hereinafter referred toas Current Protocols in Molecular Biology), Nucleic Acids Research, 10,6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315(1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl, Acad. Sci.USA, 82, 488 (1985); for example, by introducing site-directed mutationinto the DNA that codes for a polypeptide having the amino acid sequencerepresented by SEQ ID NO:1.

The number of amino acids to be deleted, substituted or added is notspecifically defined, and may be on the level of deletion, substitutionor addition attainable in a known method such as the site-directedmutagenesis as mentioned above, falling within a range of from 1 to tensof amino acids, preferably from 1 to 20, more preferably from 1 to 10,even more preferably from 1 to 5 amino acids.

In order that the polypeptide of the present invention hasN-acetylglucosamine-1-phosphate uridyltransferase activity, it isdesirable that its amino acid sequence is at least 60%, generally atleast 80%, especially at least 95. % homologous to the amino acidsequence represented by SEQ ID NO: 1, calculated according to BLAST (J.Mol. Biol., 215, 403 (1990) or FASTA [Methods in Enzymology, 183, 63-98(1990)].

However, the polypeptide of the present invention does not include knownones.

-   -   (3) A DNA coding for the polypeptide of above (1) or (2).    -   (4) A DNA comprising the nucleotide sequence represented by SEQ        ID NO:2.    -   (5) A DNA hybridizing with the DNA of above (3) or (4) under        stringent conditions, and coding for a polypeptide having        N-acetylglucosamine-1-phosphate uridyltransferase activity.

The “DNA hybridizing under stringent conditions” means that it isprepared through colony hybridization, plaque hybridization or southernhybridization using the DNA having the nucleotide sequence representedby SEQ ID NO: 2 as a probe. Concretely, for example, DNA is preparedthrough hybridization at 65° C. on a filter on which a colony orplaque-derived DNA is fixed, in the presence of from 0.7 to 1.0 mol/l ofNaCl, followed by washing the filter at 65° C. with an SSC(saline-sodium citrate) solution of about 0.1 to 2-fold concentration(the SSC solution of 1-fold concentration comprises 150 mmol/l of sodiumchloride and 15 mmol/l of sodium citrate) to thereby identify thethus-hybridized DNA.

The hybridization may be conducted according to the method described inlaboratory manuals of, for example, Molecular Cloning, 2nd Ed.; CurrentProtocols in Molecular Biology; and DNA Cloning 1: Core Techniques, APractical Approach, Second Edition, Oxford University (1995).Concretely, the hybridizable DNA is, for example, a DNA of which thenucleotide sequence is at least 60%, preferably at least 80%, morepreferably at least 95% homologous to the nucleotide sequencerepresented by SEQ ID NO:2, calculated according to BLAST or FASTA.

However, the DNA of the present invention does not include known ones.

-   -   (6) The DNA of any one of above (3) to (5), which is derived        from a microorganism belonging to the genus Corynebacterium.    -   (7) The DNA of above (6), wherein the microorganisms belonging        to the genus Corynebacterium are those of Corynebacterium        glutamicum.    -   (8) A recombinant DNA obtained by ligating any one of DNA of        above (3) to (7) with a vector.    -   (9) A transformant obtained by introducing the recombinant DNA        of above (8) into a host cell.    -   (10) The transformant of above (9), wherein the transformant        belongs to the species Corynebacterium glutamicum.    -   (11) A transformant, Corynebacterium glutamicum LS6/PV11 (FERM        BP-6937).    -   (12) A process for producing a polypeptide having        N-acetylglucosamine-1-phosphate uridyltransferase activity,        which comprises culturing the transformant of any one of        above (9) to (11) in a medium to thereby produce and accumulate        a polypeptide having N-acetylglucosamine-1-phosphate        uridyltransferase activity in a culture, and recovering the        polypeptide from the culture.    -   (13) A method for producing uridine        5′-diphosphate-N-acetylglucosamine, which comprises allowing a        culture obtained by culturing the transformant of any one of        above (9) to (11) in a medium or a treated product of a culture,        as an enzyme source, to coexist with a substrate selected from    -   (a) uridine 5′-triphosphate, glucosamine phosphates and acetyl        coenzyme A, and    -   (b) uridine 5′-triphosphate and N-acetylglucosamine phosphates,        in an aqueous medium to thereby produce and accumulate uridine        5′-diphosphate-N-acetylglucosamine in the aqueous medium, and        recovering the uridine 5′-diphosphate-N-acetylglucosamine from        the aqueous medium.

(14) The method for producing uridine 5′-diphosphate-N-acetylglucosamineof above (13), wherein the glucosamine phosphate is selected fromglucosamine-1-phosphate or glucosamine-6-phosphate, and theN-acetylglucosamine phosphate is selected fromN-acetylglucosamine-1-phosphate or N-acetylglucosamine-6-phosphate.

The present invention is described in detail hereinafter. (1)Preparation of DNA of the present invention:

The DNA of the present invention is obtained from microorganismsbelonging to the genus Corynebacterium. Any of the microorganismsbelonging to the genus Corynebacterium may be used so long as it belongsto the genus Corynebacterium, for example, including Corynebacteriumammoniagenes, Corynebacterium callunae, and Corynebacterium glutamicum.One specific example is Corynebacterium glutamicum ATCC13032.

Microorganisms belonging to the genus Corynebacterium are cultured in aknown method [for example, according to the method described in Appl.Microbiol. Biotechnol., 39, 318 (1993)]. After the culturing, thechromosomal DNA of the microorganisms is isolated and purified in aknown method [for example, according to the method described in CurrentProtocols in Molecular Biology, or Agric. Biol. Chem., 49, 2925 (1885)].

The resulting chromosomal DNA is digested with suitable restrictionenzymes, and the DNA fragment is inserted into a vector forCorynebacterium in a known method, for example, according to thedescription given in Molecular Cloning, 2nd Ed., to construct arecombinant DNA.

Any vector can be used so long as it is autonomously replicable inmicroorganisms belonging to the genus Corynebacterium, including, forexample, pCG1 (Japanese Published Unexamined Patent Application No.134500/1982), pCG2 (Japanese Published Unexamined Patent Application No.35197/1983), pCG4, pCG11 (both in Japanese Published Unexamined PatentApplication No. 183799/1982), pCE53, pCB101 (both in Japanese PublishedUnexamined Patent Application No. 105999/1993), pCE51, pCE52, pCE53 [allin Mol. Gen. Genet., 196, 175 (1984)], pAJ1844 (Japanese PublishedUnexamined Patent Application No. 21619/1983), pHK4 (Japanese PublishedUnexamined Patent Application No. 20399/1995), pHK1519 [Agric. Biol.Chem., 48, 2901 (1985)], pCV35, pECM1 [both in J. Bacteriol. 172, 1663(1990)], and pC2 [Plasmid, 36, 62 (1996)].

The recombinant DNA constructed as above is introduced intolysozyme-sensitive microorganisms of the species Corynebacteriumglutamicum. As the lysozyme-sensitive microorganisms of the speciesCorynebacterium glutamicum, any of wild type strains or mutant strainscan be used, so long as it belongs to the species Corynebacteriumglutamicum and is sensitive to lysozyme. In general, the growth of mostof wild type strains is not influenced at all by existence of lysozymeat a concentration of 100 μg/ml in a culture medium. Therefore,lysozyme-sensitive mutants are preferred for use herein.

In the present invention, the lysozyme-sensitive microorganisms meanmicroorganisms of which the growth is inhibited when a lowconcentration, at most 50 μg/ml of lysozyme exists in a culture medium.

The lysozyme-sensitive microorganisms can be derived, as a mutant, froma parent strain, of the species Corynebacterium glutamicum according toa known method (Japanese Published Examined Application Nos. 49038/1987,29555/1989, Japanese Published Unexamined Patent Application No.56678/1983). The mutant includes, for example, Corynebacteriumglutamicum ATCC31834 derived from Corynebacterium glutamicum ATCC31833(Japanese Published Unexamined Patent Application No. 56678/1983), andCorynebacterium glutamicum LS6 mentioned below, derived fromCorynebacterium glutamicum ATCC13032.

Some lysozyme-sensitive microorganisms are also sensitive to atemperature in their growth. The lysozyme-sensitive andtemperature-sensitive (hereinafter referred to aslysozyme/temperature-sensitive) microorganisms cannot grow at hightemperatures (for example, at from 34 to 39° C.) even when no lysozymeexists in a culture medium.

The mutant of this type includes, for example, Corynebacteriumglutamicum LS6 mentioned below, derived from Corynebacterium glutamicumATCC13032.

The DNA of the present invention can be obtained as a DNA thatcomplements the temperature sensitivity of thelysozyme/temperature-sensitive strain.

That is, the DNA obtained as above is ligated with a vector, and thelysozyme/temperature-sensitive strain is transformed with theDNA-ligated vector. The transformant is cultured in a lysozyme-freemedium at a temperature at which the lysozyme-sensitive microorganismscould not grow, for example, at 34 to 39° C., preferably at 36 to 38° C.The strain capable of growing in the temperature condition is selectedas that having the intended DNA, and the DNA is obtained from thestrain.

The method is described in detail below.

For the recombinant DNA introduction, any method of introducing a DNAinto the above-mentioned lysozyme/temperature-sensitive microorganismscan be employed. For example, the protoplast method [Japanese PublishedUnexamined Patent Application Nos. 186492/1982 and 56678/1983, J.Bacteriol., 159, 306 (1984)], and the electroporation method (JapanesePublished Unexamined Patent Application No. 207791/1990) are mentioned.

Alternatively, using a chromosomal DNA library of lysozyme-insensitivestrains belonging to the genus Corynebacterium as constructed inEscherichia coli, the recombinant DNA may be introduced into thelysozyme/temperature-sensitive strain of Corynebacterium glutamicumthrough bacterial conjugation transfer from Escherichia coli in thelibrary according to a known method [J. Bacteriol., 172, 1663 (1990), J.Bacteriol., 178, 5768 (1996)].

The lysozyme/temperature-sensitive strain of Corynebacterium glutamicumwith the recombinant DNA introduced thereinto is cultured, for example,in an LB medium [10 g/l of bactotryptone (produced by Difco), 5 g/l ofyeast extract (produced by Difco), 5 g/l of sodium chloride (pH 7.2)] ata high temperature (34 to 39° C.) for 24 to 72 hours. After culturing,the strain having grown in the medium is selected as that having theintended DNA.

The DNA thus obtained is, directly as it is or after digestion withsuitable restriction enzymes, ligated with a vector in an ordinarymanner, and the nucleotide sequence of the DNA is determined in anordinary sequencing method, for example, according to a dideoxy methodusing a DNA sequencer of 373A Model (produced by Parkin Elmer) [Proc.Natl. Acad. Sci. USA, 74, 5463 (1977)].

The vector to be ligated with the DNA includes, for example, pBluescriptKS(+) (produced by Stratagene), pDIRECT [Nucleic Acids Research, 18,6069(1990)], pCR-Script Amp SK(+) (produced by Stratagene), pT7Blue(produced by Novagen), pCR II (produced by Invitrogen), pCR-TRAP(produced by Gene Hunter) and pNoTAT7 (produced by 5 Prime→3 Prime).

The DNA comprising a novel nucleotide sequence obtained in the manner asabove, for example, the DNA having the sequence represented by SEQ IDNO: 2 is mentioned.

The DNA comprising the nucleotide sequence represented by SEQ ID NO: 2codes for the polypeptide having the amino acid sequence represented bySEQ ID NO: 1.

The strain that carries the plasmid comprising the DNA comprising thenucleotide sequence represented by SEQ ID NO: 2 includes, for example,Corynebacterium glutamicum LS6/pV5, and Corynebacterium glutamicumLS6/pV11.

Using a primer prepared on the basis of the nucleotide sequencedetermined as above and using the chromosomal DNA as a template, theintended DNA can be obtained through the PCR method [PCR Protocols,Academic Press (1990)].

Further, on the basis of the determined DNA sequence, the intended DNAcan also be prepared through chemical synthesis in a DNA synthesizer,such as Perceptive Biosystems' 8905 Model. (2) Preparation ofPolypeptide of the present invention:

The polypeptide of the present invention can be produced by expressingthe DNA of the present invention in host cells, according to the methoddescribed in Molecular Cloning, 2nd Ed., and Current Protocols inMolecular Biology, for example, according to the method mentioned below.

That is, a recombinant DNA is constructed by ligating the DNA of thepresent invention downstream the promoter of a suitable expressionvector, and this is introduced into host cells suitable for theexpression vector, whereby a transformant being capable of producing thepolypeptide of the present invention can be obtained. Any host cell canbe used so long as it is capable of expressing the intended gene,including, for example, bacteria, yeast cells, animal cells, insectcells and plant cells. The expression vector needs to be capable ofreplicating in host cells or being integrated with the chromosometherein, and need to have a promoter in the site in which the DNA codingfor the polypeptide of the present invention can be transcribed.

In case where prokaryotes such as bacteria are used for host cells, itis desirable that the recombinant DNA containing the DNA coding for thepolypeptide of the present invention is self-replicable in prokaryotesand comprises a promoter, a ribosome-binding sequence, the DNA thatcodes for the polypeptide of the present invention and a transcriptiontermination sequence. If desired, the recombinant DNA may contain a genefor regulating the promoter.

The expression vector includes, for example, pC2 [Plasmid, 36, 62(1996)], pBTrp2, pBTac1, pBTac2 (all commercial products of BoehringerMannheim), pKK233-2 (produced by Pharmacia), pSE280 (produced byInvitrogen), pGEMEX-1 (produced by Promega), pQE-8 (produced by QIAGEN),pKYP10(Japanese Published Unexamined Patent Application No.110600/1983), pKYP200 [Agricultural Biological Chemistry, 48, 669(1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl.Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(−) (produced byStratagene), pTrs30[prepared from Escherichia coli JM109/pTrS30(FERMBP-5407)], pTrs32 [prepared from Escherichia coli JM109/pTrS32(FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERMB-400), Japanese Published Unexamined Patent Application No.221091/1985], pGKA2 [prepared from Escherichia coli IGKA2 (FERMBP-6798), Japanese Published Unexamined Patent Application No.221091/1985], pTerm2 (U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094,U.S. Pat. No. 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J.Bacteriol., 172, 2392 (1990)], pGEX (produced by Pharmacia), and pETsystem (produced by Novagen).

For host microorganisms belonging to the genus Corynebacterium, vectorssuch as pCG1, pCG2, pCG4, pCG 11, pCE53, pCB101, pCE51, pCE52, pCE53,pAJ1844, pHK4, pHM1519, pCV35 and pECM1 are further usable in additionto the vectors mentioned above.

As the promoter to be used herein, any promoter can be used, so long asit is capable of being expressed in the host cells. For example, it isderived from Escherichia coli or phages, including trp promoter(P_(trp)), lac promoter, P_(L) promoter, P_(R) promoter and T7 promoter.Artificially-designed or modified promoters, such as a promoter of twoP_(trp)' S linked in tandem (P_(trp)×2), tac promoter, lacT7 promoter,and letI promoter, can also be used.

Plasmids which are specifically so designed that the ribosome-bindingsequence, Shine-Dalgarno sequence is spaced from the initiation codon bya suitable distance (e.g., by 6 to 18 bases) therein are preferred foruse herein.

Some nucleotides in the part of the nucleotide sequence that codes forthe polypeptide of the present invention may be substituted with anyothers so that the resulting codon may be the most suitable forpolypeptide expression in host cells, whereby the productivity of theintended polypeptide can be increased.

The recombinant vector for use in the present invention does not alwaysrequire a transcription termination sequence for expression of the DNAof the present invention. In the recombinant vector, however, it isdesirable that a transcription termination sequence is just downstreamthe structural gene.

The host cells include microorganism belonging to the genus Escherichia,Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium andPseudomonas and the like, including, for example, Escherichia coliXL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichiacoli MC1000, Escherichia coli KY3276, Escherichia coli W1485,Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49,Escherichia coli W3110, Escherichia coli NY49, Serratia ficaria,Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillussubtilis, Bacillus amyloliquefaciens, Brevibacterium ammoniagenes,Brevibacterium immariophilum ATCC 14068, Brevibacterium saccharolyticumATCC 14066, Brevibacterium flavum ATCC 14067, Brevibacteriumlactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 13032,Corynebacterium acetoacidophilum ATCC 13870, Microbacteriumammoniaphilum ATCC 15354, and Pseudomonas sp. D-0110.

For introducing the recombinant vector into the host cells, any methodof introducing DNA thereinto can be employed. For example, the methodassisted by calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)],the protoplast method (Japanese Published Unexamined Patent ApplicationNo. 248394/19B8), the electroporation method (Japanese PublishedUnexamined Patent Application No. 207791/1990), and the methodsdescribed in Gene, 17, 107 (1982) and Molecular & General Genetics, 168,111 (1979) are mentioned.

In case where yeast is used for host cells, the expression vector forthis purpose includes, for example, YEp13 (ATCC 37115), YEp24 (ATCC37051) and YCp50(ATCC 37419).

As the suitable promoter, any promoter can be used, so long as it iscapable of being expressed in yeast cells, including, for example,promoters for genes participating in glycolysis such as hexosekinase, aswell as PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1promoter, gal 10 promoter, heat-shock polypeptide promoter, MFα1promoter, and CUP1 promoter.

The host cells include a microorganism belonging to the genusSaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces and the like,including, for example, Saccharomyces cerevisiae, Schizosaccharomycespombe, Kluyveromyces lactis, Trichosporon pullulans and Schwanniomycesalluvius.

For introducing the recombinant DNA into the host cells, any method ofintroducing DNA into yeast cells is employable. For example, theelectroporation method [Methods Enzymol., 194, 182 (1990)], thespheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)1, thelithium acetate method [J. Bacteriol., 153, 163 (1983)], and the methoddescribed in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) are mentioned.

In case where animal cells are used for host cells, the expressionvector for this purpose includes, for example, pcDNAI, pcDM8(commercially sold by Funakoshi), pAGE107 [Japanese Published UnexaminedPatent Application No. 22979/1991, Cytotechnology, 3, 133 (1990)],pAS3-3 (Japanese Published Unexamined Patent Application No.227075/1990), pCDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp (produced byInvitrogen), pREP4 (produced by Invitrogen), pAGE103 [J. Biochemistry,101, 1307 (1987)], and pAGE210.

As the promoter, any promoter may be used, so long as it is capable ofbeing expressed in animal cells, including, for example, the promoter ofIE (immediate early) gene of cytomegalovirus (CMV), the early promoterof SV40, the promoter of retrovirus, as well as metallothioneinpromoter, heat-shock promoter, and SRα promoter. The enhancer of the IEgene of human CMV may be used together with the promoter.

The host cells include, for example, Namalwa cells of human, COS cellsof monkey, CHO cells of Chinese hamster, and HBT5637 cells (JapanesePublished Unexamined Patent Application No. 299/1988).

For introducing the recombinant vector into the animal cells, any methodof introducing DNA into animal cells is employable. For example, theelectroporation method [Cytotechnology, 3, 133 (1990)], the calciumphosphate method (Japanese Published Unexamined Patent Application No.227075/1990), and the lipofection method (Proc. Natl. Acad. Sci. USA,84, 7413 (1987)] are mentioned.

In case where insect cells are used for host cells, the polypeptide maybe expressed, for example, according to the methods described in CurrentProtocols in Molecular Biology; Baculovirus Expression Vectors, ALaboratory Manual, W. H. Freeman and Company, New York (1992); andBio/Technology, 6, 47 (1988).

That is, a recombinant gene introduction vector and a Baculovirus aresimultaneously introduced into insect cells to obtain a recombinantvirus in a culture supernatant of the insect cell, and then insect cellsare infected with the recombinant virus so as to express thepolypeptide.

The gene introduction vector to be used in the method includes, forexample, pVL1392, pVL1393 and pBlueBacIII (all produced by Invitrogen).

An example of the Baculovirus is, for example, Autographa californicanuclear polyhedrosis virus that infects armyworms of Barathra.

The insect cells include, for example, Spodoptera frugiperda oocytes,Sf9, Sf21 [Baculovirus Expression Vectors, A Laboratory Manual, W. H.Freeman and Company, New York (1992)], and Trichoplusia ni oocytes, High5 (produced by Invitrogen).

For simultaneously introducing the recombinant gene introduction vectorand the Baculovirus into insect cells to prepare a recombinant virus,for example, the calcium phosphate method (Japanese Published UnexaminedPatent Application No. 227075/1990) and the lipofection method [Proc.Natl. Acad. Sci. USA, 84, 7413 (1987)] are employable.

In case where plant cells are used for host cells, the expression vectorfor the purpose includes, for example, Ti plasmid and tobacco mosaicvirus vector.

As the promoter, any promoter can be used so long as it is capable ofbeing expressed in plant cells, including, for example, 35S promoter ofcauliflower mosaic virus (CaMV), and rice actin 1 promoter.

The plant cells for host cells include, for example, those of tobacco,potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, and barley.

For introducing the recombinant DNA into such plant cells, any method ofintroducing DNA thereinto is employable, including, for example, themethod by using Agrobacterium (Japanese Published Unexamined PatentApplication Nos. 140885/1984 and 70080/1985, w094/00977), theelectroporation method (Japanese Published Unexamined Patent ApplicationNo. 251887/1985), and the method by using a particle gun (gene gun)(Japanese Published Examined Patent No. 2,606,856, 2,517,813).

The gene expression may be conducted in a mode of direct expression, orin a mode of secretion production or fused protein expression accordingto the method described in Molecular Cloning, 2nd Ed.

In case where the gene is expressed in yeast cells, animal cells, insectcells or plant cells, it gives a polypeptide with a saccharide or sugarchain added thereto.

The transformant prepared in the manner as above is cultured in amedium, and the polypeptide of the present invention is produced andaccumulated in a culture, and recovered from the culture. For culturingthe transformant of the present invention in a medium, any method forculturing a host cell as generally used in the art is employable.

In case where the transformant of the present invention is prepared bythe use of bacteria such as Escherichia coli or eukaryotic host cellsincluding, for example, yeast cells, the medium in which thetransformant is cultured may be any natural or synthetic mediumcontaining carbon sources, nitrogen sources and inorganic salts whichcan be assimilated by the transformant and in which the transformant canbe efficiently cultured.

The carbon sources may be any ones which can be assimilated by thetransformant, including, for example, carbohydrates such as glucose,fructose, sucrose, molasses containing them, starch or starchhydrolyzates; organic acids such as acetic acid and propionic acid; andalcohols such as ethanol and propanol.

The nitrogen sources include, for example, ammonia, ammonium salts ofinorganic acids and organic acids, such as ammonium chloride, ammoniumsulfate, ammonium acetate, ammonium phosphate; other nitrogen-containingcompounds; and peptone, meat extracts, yeast extracts, corn steepliquor, casein hydrolyzates, soy bean meal, soy bean meal hydrolyzates,various cells obtained by fermentation and their digested products.

The inorganic salts include, for example, potassium dihydrogenphosphate,dipotassium hydrogenphosphate, magnesium phosphate, magnesium sulfate,sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, andcalcium carbonate.

Culturing the transformant in the medium is generally conducted underaerobic conditions, for example, by shaking culture or submerged spinnerculture under aeration. The culturing temperature is preferably from 15to 40° C., and the culturing period is generally from 16 hours to 7days. Preferably, the pH of the culture is from 3.0 to 9.0. For the pHcontrol, any of inorganic or organic acids, alkali solutions, urea,calcium carbonate or ammonia or the like may be used.

If desired, antibiotics such as ampicillin, tetracycline and kanamycinmay be added to the medium in which the transformant is cultured.

In case where microorganisms transformed with a recombinant vectorhaving an inducible promoter are cultured, an inducer may be added tothe medium, if desired. For example, when microorganisms transformedwith a recombinant vector having lac promoter are cultured,isopropyl-β-D-thiogalactopyranoside may be added to the medium; and whenmicroorganisms transformed with a recombinant vector having trp promoterare cultured, indole-acrylic acid may be added to the medium.

The medium in which the transformant prepared by using animal cells as ahost cell is cultured may be any ordinary one, including, for example,RPMI1640 medium [The Journal of the American Medical Association, 199,519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco'smodified MEM medium [Virology, 8, 396(1959)], 199 medium [Proceeding ofthe Society for the Biological Medicine, 73, 1 (1950)], and those mediawith fetal calf serum therein.

Culturing the transformant in the medium is conducted generally at pH 6to 8, at 30 to 40° C. in the presence of 5% CO₂ for 1 to 7 days.

If desired, antibiotics such as ampicillin and tetracycline may be addedto the medium in which the transformant is cultured.

The medium in which the transformant prepared by using insect cells as ahost cell is cultured may be any ordinary one, including, for example,TNM-FH medium (produced by PharMingen), Sf-900 II SFM medium (producedby Life Technologies), ExCell 400, ExCell 405 [both produced by JRHBiosciences] and Grace's Insect Medium [Nature, 195, 788 (1962)].

Culturing the transformant in the medium is conducted generally at pH 6to 7, at 25 to 30° C. for 1 to 5 days.

If desired, antibiotics such as gentamycin may be added to the medium inwhich the transformant is cultured.

The transformant cells prepared by using plant cells as a host cell maybe cultured as they are, or after differentiated into plant cells ororgans. The medium in which the transformant is cultured may be anyordinary one, including, for example, Murashige & Skoog (MS) medium,White medium, and those media with a plant hormone such as auxin orcytokinin therein.

Culturing the transformant in the medium is conducted generally at pH 5to 9, at 20 to 40° C. for 3 to 60 days.

If desired, antibiotics such as kanamycin and hygromycin may be added tothe medium in which the transformant is cultured.

As described above, the transformant derived from microorganisms, animalcells or plant cells carrying a recombinant vector ligated with the DNAcoding for the polypeptide of the present invention is cultured in anordinary manner to thereby produce and accumulate the polypeptide, andthe polypeptide is recovered from the culture.

The gene expression may be conducted in a mode of direct expression, orin a mode of secretion production or fused polypeptide expressionaccording to the method described in Molecular Cloning, 2nd Ed.

The method for producing the polypeptide of the present inventionincludes intracellular production, extracellular secretion or productionon cell outer membrane of host cells, and the method can be selecteddepending on the host cells used or on alteration the structure of thepolypeptide to be produced.

In case where the polypeptide of the present invention is producedinside host cells or on the outer membrane of host cells, it can besecreted in the extracellular portion from the host cells, according tothe method of Paulson, et al. [J. Biol. Chem., 264, 17619 (1989)], themethod of Lowe, et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989),Genes Develop., 4, 1288 (1990)] or the methods described in JapanesePublished Unexamined Patent Application No. 336963/1993 and WO94/23021.

That is, the polypeptide of the present invention can be secreted in theextracellular portion from the host cells, by expressing it in the formof a polypeptide containing the active site of the polypeptide of thepresent invention and having a signal peptide upstream it, according togene recombination technology.

The yield of the polypeptide to be produced can be increased in a geneamplification system using a dihydrofolate reductase gene or the like,according to the method described in Japanese Published UnexaminedPatent Application No. 227075/1990.

In addition, the gene-introduced animal or plant cells may bere-differentiated to construct gene-introduced animal individuals(transgenic non-human animals) or plant individuals (transgenic plants).Using these individuals, the polypeptide of the present invention may beproduced.

In case where the transformant is an animal individual or plantindividual, it may be raised or cultivated in an ordinary manner tothereby produce and accumulate the intended polypeptide therein, and thepolypeptide is recovered from the animal or plant individual.

For producing the polypeptide of the present invention in animalindividuals, for example, an animal is transformed with the gene codingfor the polypeptide, and the polypeptide is produced in the transformantanimal according to known methods [American Journal of ClinicalNutrition, 63, 639S (1996), American Journal of Clinical Nutrition, 63,627S (1996), Bio/Technology, 9, 830 (1991)].

For animal individuals, for example, the transgenic non-human animalscarrying the DNA coding for the polypeptide of the present invention areraised to thereby produce and accumulate the polypeptide in the animals,and the polypeptide is recovered from the animals. The site of theanimals in which the polypeptide is produced and accumulate is, forexample, milk (Japanese Published Unexamined Patent Application No.309192/1988), eggs of the animals, and the like. The promoter to be usedmay be any promoter which is capable of being expressed in animals. Forexample, mammary gland cell-specific promoters such as α-caseinpromoter, β-casein promoter, ⊕-lactoglobulin promoter, whey acidicprotein promoter, and the like, are preferably used.

For producing the polypeptide of the present invention in plantindividuals, for example, a transgenic plant carrying the DNA coding forthe polypeptide of the present invention is cultivated in known methods[Tissue Culture, 20 (1994), Tissue Culture, 21 (1995), Trends inBiotechnology, 15, 45 (1997)] to thereby produce and accumulate thepolypeptide in the plant, and the polypeptide is recovered from theplant.

For isolating and purifying the polypeptide having been expressedaccording to the methods mentioned above, from the transformantcultures, any ordinary enzyme isolation and purification method isemployable.

For example, when the polypeptide of the present invention is expressedin soluble forms inside the transformant cells, the cells are, aftercultured, recovered from the culture by centrifuging the culture, thensuspended in an aqueous buffer, and disrupted with an ultrasonicdisrupter, French Press, Manton-Gaulin homogenizer, Dynomill or the liketo obtain a cell-free extract. The cell-free extract is centrifuged, andthe resulting supernatant is purified through ordinary enzyme isolationand purification methods. Specifically, for example, the supernatant ispurified through solvent extraction, salting-out or desalting withsulfate ammonium or the like, precipitation with organic solvent,anion-exchange chromatography on resin such as diethylaminoethyl(DEAE)-Sepharose or DIAION HPA-75 (produced by Mitsubishi ChemicalIndustries), or the like, cation-exchange chromatography on resin suchas S-Sepharose FF (produced by Pharmacia), hydrophobic chromatography onresin such as butyl Sepharose or phenyl Sepharose, gel filtrationthrough molecular sieve, affinity chromatography, chromatofocusing, orelectrophoresis such as isoelectric focusing, or the like. Thepurification methods may be used either singly or as combined to obtainthe intended pure product.

In case where the polypeptide is expressed as an inclusion body in thecells, the cells are similarly recovered, disrupted and centrifuged togive a precipitated fraction that contains the inclusion body of thepolypeptide. The thus-recovered inclusion body of the polypeptide issolubilized with a protein denaturing agent. The solubilized solution isthen diluted or dialyzed to thereby lower the concentration of theprotein denaturing agent in the solution, in which concentration, theprotein may not be denatured. Through the process, the solubilizedpolypeptide is renatured to have its own normal tertiary structure.After thus processed, the polypeptide is purified through the sameisolation and purification as above to be a pure product.

In case where the polypeptide of the present invention or thepolypeptide derivative with a sugar chain added thereto is secreted fromthe cells, the polypeptide or the sugar chain-added polypeptidederivative can be recovered in the culture supernatant. Specifically,the culture is centrifuged in the same manner as above to obtain theculture supernatant containing a soluble fraction, and the fraction ispurified through the same isolation and purification as above to obtaina pure polypeptide product.

As the polypeptide thus obtained in the manner as above, for example,the polypeptide having the amino acid sequence represented by SEQ IDNO:1 is mentioned.

The polypeptide of the present invention can be produced throughchemical synthesis of, for example, an Fmoc method(fluorenylmethyloxycarbonyl method) or a tBoc method (t-butyloxycarbonylmethod). It may also be produced through chemical synthesis usingpeptide synthesizers such as those produced by Advanced ChemTech,Parkin-Elmer, Pharmacia, Protein Technology Instrument, Synthecell-vega,PerSeptive, and Shimadzu.

(3) Preparation of UDP-GlcNAc:

Using the transformant culture obtained in the above (2) or its treatedproduct obtained by treating the culture in various methods, as anenzyme source, and putting the enzyme source and a substrate for it inan aqueous medium, UDP-GlcNAc can be produced in the aqueous medium.

The treated product of the transformant culture includes, for example,concentrated products of the culture, dried products of the culture,cells obtained by centrifuging the culture, dried products of the cells,lyophilized products of the cells, surfactant-treated products of thecells, ultrasonically-treated products of the cells,mechanically-disrupted products of the cells, solvent-treated productsof the cells, enzyme-treated products of the cells, polypeptidefractions from the cells, immobilized products of the cells, and enzymeproducts extracted from the cells.

The concentration of the enzyme source to be used in producingUDP-GlcNAc may be from 1 to 500 g wet cells/l, but preferably from 10 to300 g wet cells/l, in terms of the weight of the cells (wet cells) justafter centrifugation of the culture.

The aqueous medium to be used in producing the UDP-GlcNAc includes, forexample, water; buffers such as phosphate, carbonate, acetate, borate,citrate, Tris; alcohols such as methanol, ethanol; esters such as ethylacetate; ketones such as acetone; and amides such as acetamide. Theculture of the microorganisms serving as the enzyme source may also beused for the aqueous medium.

If desired, a surfactant or organic solvent may be added to the aqueousmedium for producing UDP-GlcNAc. As the surfactant, any surfactant maybe used so long as it does not inhibit with the production ofUDP-GlcNAc, including, for example, nonionic surfactants such aspolyoxyethylene-octadecylamine (e.g., Nymeen S-215, produced by NipponOil & Fats); cationic surfactants such as cetyltrimethylammonium bromideand alkyldimethylbenzylammonium chlorides (e.g., Cation F2-40E, producedby Nippon Oil & Fats); anionic surfactants such as lauroyl sarcosinate;and tertiary amines such as alkyldimethylamines (e.g., Tertiary AmineFB, produced by Nippon Oil & Fats). One or more of such surfactants maybe used either singly or as combined. The surfactant concentration isgenerally from 0.1 to 50 g/l. The organic solvent includes, for example,xylene, toluene, aliphatic alcohols, acetone, and ethyl acetate. Thesolvent concentration is generally from 0.1 to 50 ml/l.

The substrate to be used in producing UDP-GlcNAc includes uridine5′-triphosphate and glucosamine derivatives.

The glucosamine derivatives are, for example, glucosamine phosphatesselected from glucosamine-1-phosphate and glucosamine-6-phosphate, orN-acetylglucosamine phosphates selected fromN-acetylglucosamine-1-phosphate and N-acetylglucosamine-6-phosphate. Incase where a glucosamine phosphate is used as the glucosaminederivative, acetyl coenzyme A (acetyl CoA) need to be added thereto.However, when acetyl CoA already exists in the enzyme source, it may notbe added to the aqueous medium.

In case where the enzyme product obtained through cell extraction isused for the enzyme source, the glucosamine derivative to be used withthe enzyme product is glucosamine-1-phosphate orN-acetylglucosamine-1-phosphate. However, when the enzyme sourcecontains an enzyme having the activity to convertglucosamine-6-phosphate or N-acetylglucosamine-6-phosphate intoglucosamine-1-phosphate or N-acetylglyucosamine-1-phsophate,glucosamine-6-phosphate or N-acetylglucosamine-6-phosphate may be usedas the glucosamine derivative.

The substrate concentration may be from 0.1 to 500 mmol/l.

The reaction in the aqueous medium to produce UDP-GlcNAc is conducted atpH 5 to 10, preferably from 6 to 8, at 20 to 60° C. for 1 to 96 hours.If desired, an inorganic salt such as MgCl₂ may be added to the reactionmixture.

UDP-GlcNAc produced in the aqueous medium can be quantified in a knownmanner such as HPLC [for example, as in WO98/12343].

UDP-GlcNAc produced in the reaction mixture can be recovered in anyordinary manner using, for example, activated charcoal or ion-exchangeresin.

Examples of the present invention are described below, to which,however, the present invention is not limited.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the result of a temperature-sensitivity complementationtest with Corynebacterium glutamicum LS6, in which various deletionplasmids having a ligated DNA fragment of about 7 kb were prepared andtested. “Plac” indicates the site of the lactose promoter existing inthe vector pC2. “+”indicates that the plasmid complemented thetemperature sensitivity of the transformant. An outline of therestriction enzyme map of the DNA fragment insert of about 7 kb, and thesite and the direction of the glmU gene are shown in FIG. 1.

BEST MODES OF CARRYING OUT THE INVENTION EXAMPLE 1 Preparation ofChromosomal DNA of Corynebacterium glutamicum ATCC 13032

Corynebacterium glutamicum ATCC 13032 was inoculated in 10 ml of an L′medium (1% of polypeptone, 0.5% of yeast extract, 0.5% of sodiumchloride, 0.1% of glucose, 20 mg/l of thiamine hydrochloride, pH 7.2)and cultured overnight at 30° C.

After culturing, the cells were recovered from the culture throughcentrifugation.

The cells were washed with TE buffer [10 mmol/l of Tris-HCl, 1 mol/l ofethylenediaminetetraacetate (EDTA), pH 8.0], and suspended in 800 μl ofthe same buffer. 40 μl of a lysozyme solution (50 mg/ml) and 20 μl of anRNase A solution (10 mg/ml) were added to the suspension, and reacted at37° C. for 1 hour.

20 μl of sodium dodecyl sulfate (SDS) solution (20%) was added to thereaction mixture, and reacted at 70° C. for 1 hour. 24 μl of aproteinase K solution (20 mg/ml) was added thereto and reacted at 50° C.for 1 hour. 24 μL of the proteinase K solution was further addedthereto, and reacted at 50° C. for 1 hour. To the reaction solution,phenol of the same amount as that of the reaction solution was added andstirred, and then left at 4° C. overnight, whereby the DNA was extractedout in the aqueous phase, and the aqueous phase was collected.

To the aqueous phase, phenol/chloroform (1/1, vol/vol) of the sameamount as that of the aqueous phase was added, stirred, and extractedfor 2 hours, and the aqueous phase was recovered. To the aqueous phase,chloroform/isoamyl alcohol (24/1, vol/vol) of the same amount as that ofthe aqueous phase was added, stirred, and extracted for 30 minutes, andthe aqueous phase was recovered. To the aqueous phase, ethanol of twiceof the aqueous phase was added, and the DNA was precipitated. Theresulting precipitate was dissolved in 300 μl of a TE buffer, and usedas the chromosomal DNA below.

EXAMPLE 2 Separation of Temperature-Sensitivity Complementing Gene

0.5 μg of the chromosomal DNA obtained in Example 1 and 0.5 μg of aplasmid pC2 were digested with BamHI, and their fragments were ligatedusing a ligation kit (Takara DNA Ligation Kit ver. 2, produced byTAKARA), at 16° C. for 16 hours.

Lysozyme/temperature-sensitive Corynebacterium glutamicum LS6 wastransformed with the ligated product by electroporation (JapanesePublished Unexamined Patent Application No. 207791/1990), and thetransformant was selected based on the property of the strain LS6 whichis sensitive to temperature. That is, the transformant was spread on anL′-agar plate medium (prepared by adding 1.5% agar to an L′-medium)containing 5 μl/ml kanamycin, and cultured at 37° C. for 3 days.

The resulting colonies were cultured according to the method of Example1, and the plasmid was recovered according to the method described inMolecular Cloning, 2nd Ed. The recovered plasmid pV5 was analyzed forits structure. It was found that the plasmid pV5 has a DNA fragment ofabout 7 kb derived from Corynebacterium glutamicum inserted into theBamHI site of the plasmid pC2.

Various deletion plasmids were obtained by using the DNA fragment ofabout 7 kb inserted into the plasmid pV5, in an ordinary manner. Witheach deletion plasmid, Corynebacterium glutamicum LS6 was transformed,and the temperature sensitivity of the transformant was investigated. Itwas found that a BamHI-SacI fragment of 2.3 kb was required tocomplement the temperature sensitivity (FIG. 1).

The nucleotide sequence of the BamHI-SacI fragment of 2.3 kb wasdetermined, and it was confirmed that an open reading frame (ORF) of1455 bp having the nucleotide sequence represented by SEQ ID NO: 2 andcoding for the amino acid sequence of 485 amino acid residuesrepresented by SEQ ID NO; 1 exists in the region of this fragment.

The amino acid sequence homology of the DNA to different amino acidsequences was studied according to BLAST P2.0.10. As a result, the aminoacid sequence represented by SEQ ID NO: 1 was 40% homologous to theamino acid sequence of GlmU derived from Escherichia coli heretoforereported [J. Biochem., 115, 965 (1994)], and was 40% homologous to theamino acid sequence of GlmU derived from Bacillus subtilis [J. Gen.icrobiol., 139, 3185 (1993)].

The nucleotide sequence homology of the DNA to different nucleotidesequences was also studied according to BLAST N2.0.10. As a result, nohomology of the nucleotide sequence represented by SEQ ID NO: 2 thatcodes for the amino acid sequence was found to any others.

Corynebacterium glutamicum LS6/pV11 that contains the plasmid pv11having the BamHI-SacI fragment of 2.3 kb was deposited as FERM BP-6937on Nov. 12, 1999 with the International Patent Organism Depositary,National Institute of advanced Industrial Science and Technology (AISTTsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaragi-ken305-8566 Japan) [National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology, 1-3, Higashi 1-Chome,Tsukuba city, Ibaragi prefecture, 305-8566, Japan].

EXAMPLE 3 Determination of GlmU Activity

Corynebacterium glutamicum LS6/pV11 obtained in Example 2 was culturedaccording to the method of Example 1, and the culture was centrifuged toobtain wet cells. If desired, the wet cells can be stored at −20° C.,and the frozen cells can be thawed before use.

In 0.2 ml of a mixture comprising 100 mmol/l of Tris-HCl (pH 8.0), 10mmol/l of glucosamine-1-phosphate, 10 mmol/l of acetyl CoA, 10 mmol/l ofUTP, 2 mmol/l of MgCl₂, 4 g/l of Nymeen S-215 and 10 ml/l of xylene, and10 g/l of the wet cells, the reaction was conducted at 37° C. for 10minutes.

After the reaction was carried out, the reaction mixture was analyzedfor UDP-GlcNAc formed therein, according to the method described inWO98/12343, and it was confirmed that 7.4 mmol/l of UDP-GlcNAc wasproduced and accumulated in the reaction mixture. On the other hand, thestrain LS6/pC2 containing the vector alone produced 1.2 mmol/l ofUDP-GlcNAc.

INDUSTRIAL APPLICABILITY

According to the present invention, mass-scale production of the GlmUpolypeptide derived from microorganisms belonging to the genusCorynebacterium glutamicum has been enabled by genetic recombinanttechnology. Using the enzyme, UDP-GlcNAc can be produced efficiently.

1. An isolated or purified polypeptide comprising the amino acidsequence represented by SEQ ID NO:
 1. 2. An isolated or purified DNAcoding for the polypeptide of claim
 1. 3. An isolated or purified DNAcomprising the nucleotide sequence represented by SEQ ID NO:2.
 4. Arecombinant DNA obtained by ligating the DNA of claim 2 or 3 with avector.
 5. A transformant obtained by introducing the recombinant DNA ofclaim 4 into a host cell.
 6. The transformant of claim 5, wherein thehost cell is Corynebacterium glutamicum.
 7. A process for producing apolypeptide having N-acetylglucosamine-1-phosphate uridyltransferaseactivity, which comprises culturing the transformant of claim 5 in amedium to produce and accumulate a polypeptide havingN-acetylglucosamine-1-phosphate uridyltransferase activity in a culture,and recovering the polypeptide from the culture.
 8. A method forproducing uridine 5′-diphosphate-N-acetylglucosamine, which comprisescontacting a culture of the transformant or immobilized cells, of claim5 with uridine 5′-triphosphate and N-acetylglucosamine phosphate, in anaqueous medium, to produce and accumulate uridine5′-diphosphate-N-acetylglucosamine in the aqueous medium, and recoveringthe uridine 5′-diphosphate-N-acetylglucosamine from the aqueous medium.9. The method for producing uridine 5′-diphosphate-N-acetylglucosamineof claim 8, wherein the N-acetylglucosamine phosphate isN-acetylglycosamine-1-phosphate or N-acetylglucosamine-6-phosphate. 10.A method for producing uridine 5′-diphosphate-N-acetylglucosamine, whichcomprises: contacting a cell extract of the transformant of claim 5 orN-acetylglucosamine-1-phosphate uridyltransferase obtained therefromwith uridine 5′-triphosphate, glucosamine phosphate and acetyl coenzymeA, in an aqueous medium to produce and accumulate uridine5′-diphosphate-N-acetylglucosamine in the aqueous medium, and recoveringthe uridine 5′-diphosphate-N-acetylglucosamine from the aqueous medium.11. The method for producing uridine 5′-diphosphate-N-acetylglucosamineof claim 10, wherein the glucosamine phosphate isglucosamine-1-phosphate or glucosamine-6-phosphate.
 12. An isolated orpurified DNA which hybridizes with the DNA of SEQ ID NO. 2 at 65° C. ona filter on which a colony or plaque-derived DNA which is derived frommicroorganisms belonging to the genus Corynebacterium is fixed, in thepresence of from 0.7 to 1.0 mol/l NaCl, followed by washing at 65° C.with 0.1×SSC, and which codes for a polypeptide havingN-acetylglucosamine-1-phosphate uridyltransferase activity.
 13. The DNAof claim 12, wherein the Corynebacterium is Corynebacterium glutamicum.14. A recombinant DNA obtained by ligating the DNA of claim 12 with avector.
 15. A transformant obtained by introducing the recombinant DNAof claim 14 into a host cell.
 16. The transformant of claim 15, whereinthe host cell is Corynebacterium glutamicum.
 17. A process for producinga polypeptide having N-acetylglucosamine-1-phosphate uridyltransferaseactivity, which comprises culturing the transformant of claim 15 in amedium to thereby produce and accumulate a polypeptide havingN-acetylglucosamine-1-phosphate uridyltransferase activity in a culture,and recovering the polypeptide from the culture.
 18. A method forproducing uridine 5′-diphosphate-N-acetylglucosamine, which comprises:contacting a culture of the transformant, or immobilized cells, of claim15 with uridine 5′-triphosphate and N-acetylglucosamine phosphate, in anaqueous medium, to produce and accumulate uridine5′-diphosphate-N-acetylglucosamine in the aqueous medium, and recoveringthe uridine 5′-diphosphate-N-acetylglucosamine from the aqueous medium.19. The method for producing uridine 5′-diphosphate-N-acetylglucosamineof claim 18, wherein the N-acetylglucosamine phosphate isN-acetylglycosamine-1-phosphate or N-acetylglucosamine-6-phosphate. 20.A method for producing 5′-diphosphate-N-acetylglucosamine, whichcomprises: contacting a cell extract of the transformant of claim 15 orN-acetylglucosamine-1-phosphate uridyltransferase obtained therefromwith uridine 5-triphosphate, glucosamine phosphate and acetyl coenzyme Ain an aqueous medium to produce and accumulate uridine5′-diphosphate-N-acetylglucosamine in the aqueous medium, and recoveringthe uridine 5′-diphosphate-N-acetylglucosamine from the aqueous medium.21. The method for producing uridine 5′-diphosphate-N-acetylglucosamineof claim 20, wherein the glucosamine phosphate isglucosamine-1-phosphate or glucosamine-6-phosphate.
 22. Corynebacteriumglutamicum LS6/PV11 (FERM BP-6937).